Feleucins: Novel Bombinin Precursor-Encoded Nonapeptide Amides from the Skin Secretion ofBombina variegata

Abstract

The first amphibian skin antimicrobial peptide (AMP) to be identified was named bombinin, reflecting its origin from the skin of the European yellow-bellied toad (Bombina variegata). Bombinins and their related peptides, the bombinin Hs, were subsequently reported from other bombinid toads. Molecular cloning of bombinin-encoding cDNAs from skin found that bombinins and bombinin Hs were coencoded on the same precursor proteins. Here, we report the molecular cloning of two novel cDNAs from a skin secretion-derived cDNA library of B. variegata whose open-reading frames each encode a novel bombinin (GIGGALLNVGKVALKGLAKGLAEHFANamide) and a C-terminally located single copy of a novel nonapeptide (FLGLLGGLLamide or FLGLIGSLLamide). These novel nonapeptides were named feleucin-BV1 and feleucin-BV2, respectively. The novel bombinin exhibited 89% identity to homologues from the toads, B. microdeladigitora and B. maxima. The feleucins exhibited no identity with any amphibian AMP archived in databases. Synthetic feleucins exhibited a weak activity against Staphylococcus aureus (128–256 mg/L) but feleucin-BV1 exhibited a synergistic action with the novel bombinin. The present report clearly demonstrates that the skin secretions of bombinid toads continue to represent a source of peptides of novel structure that could provide templates for the design of therapeutics.

Feleucin‐BO1: A Novel Antimicrobial Non‐Apeptide Amide from the Skin Secretion of the Toad, Bombina orientalis, and Design of a Potent Broad‐Spectrum Synthetic Analogue, Feleucin‐K3

“…Following successful syntheses of all three peptides using solid‐phase Fmoc chemistry, they were subjected to MIC and MBC assays, the results of which are shown in Table . As expected from previous data derived from the B. variegata feleucins , the synthetic replicate of feleucin‐BO1 was moderately effective against S. aureus with an MIC of 34 μ m and an MBC of 128 μ m . However, the cationicity‐enhanced analogue, F‐K3, exhibited a more potent and broader spectrum of action with MICs of 7 μ m against S. aureus and C. albicans and an MIC of 14 μ m against E. coli .…”

“…While considerably more effective than the native feleucin‐BO1, these values were not appreciably different from those obtained with the feleucin analogue, F‐K3. This cationicity‐enhanced analogue of feleucin‐BO1 would have had essentially the same structure if engineered from the feleucin prototype peptides, BV1 and BV2 from B. variegata . These data clearly indicate the potential of conserved natural antimicrobial peptide templates, even those with poor efficacy, for the generation of analogues with significantly enhanced potencies and broadened spectra of activities on target micro‐organisms.…”

“…Feleucin‐BO1 exhibited single residue substitutions compared to the prototypes, feleucin‐BV1 (Ser (S) for Gly (G) at position 7) and feleucin‐BV2 (Leu (L) for Ile (I) at position 5) (Figure A). The processing sites and locations of respective peptides within the common precursor were identical to those previously described for the homologous B. variegata skin proteins .…”

“…The novel feleucin described here was named feleucin‐BO1, in accordance with the nomenclature adopted for the prototype peptides from the skin secretion of B. variegata , but the novel bombinin described here was named FPA‐bombinin‐BO. The secondary structures of each peptide were predicted using online programmes and their physico‐chemical characteristics were likewise determined (Table ).…”

Discovery of two bombinin peptides with antimicrobial and anticancer activities from the skin secretion of Oriental fire‐bellied toad, Bombina orientalis

“…Afterwards, the full‐length prepropeptide nucleic acid sequence data were obtained by reverse transcription followed by 3′RACE procedures with a SMARTer RACE kit (Clontech, Mountain View, CA, USA). Briefly, 3′RACE reactions were carried out using a nest universal primer (UPM, supplied with the kit) and a sense primer (S1: 5′‐GATGAWKTTTAAGTACATARTTGCRGT‐3′) (W = A/T; K = T/G; R = A/G) that was designed to a highly conserved segment of the 5′‐untranslated region of bombinin precursor‐encoding cDNAs from Bombina species . The PCR amplification was completed using the following parameters: initial denaturation step: 94°C for 1 min; 35 cycles, in each cycle: 94°C for 30 s, 56°C for 30 s and 72°C for 3 min.…”